Storing oligos: 7 things you should know

Lab tips: Researchers often have questions about the stability of their oligos and how best to store them. Review these important considerations and supporting data, which were generated from an ongoing, multi-year, longitudinal stability study.

Properly storing your oligos when they are not in use is crucial to preserving their stability and ensuring reliable results in your experiments. Below are some useful tips for you to keep in mind. For guidance on resuspending and diluting oligos, see the article, Tips for resuspending and diluting your oligonucleotides.

For unmodified, DNA oligos:

1. Oligo stability depends on storage temperature and storage medium.

Of these 2 variables, temperature is most important. When stored at –20°C (frozen), IDT oligos remain stable for 2 years (24 months), regardless of whether they are stored dry or resuspended in TE buffer or nuclease-free water (Figure 1, Panel A).

Similarly, at 4°C (refrigerated), there is no significant difference in stability between storage mediums. At this temperature, oligos stored across the same 3 mediums—dry, TE buffer, and nuclease-free water—are stable beyond 1 year (>60 weeks; Figure 1, Panel B).

Our general recommendation is: If storing your oligos for long periods of time, store them in the freezer, as this ensures stability for the longest duration, regardless of storage medium. It is also best practice to minimize oligo exposure to UV light.

Figure 1. Oligo stability in 3 storage mediums at –20°C, 4°C, and 37°C. Stability of oligos stored in TE buffer (IDTE, pH 8.0), nuclease-free water, or dry, for up to 24 months was measured in a qPCR assay by assessing ∆Cq of the assay. (A) Oligos stored at –20°C remained intact (∆Cq >1.5) for 24 months, regardless of storage medium. (B) Oligos stored at 4°C remained intact after 60 weeks, with insignificant variability across storage mediums. (C) Oligos stored at 37°C showed dramatic differences in stability across storage mediums. Resuspension in TE buffer provided the greatest stability (~150 weeks), followed by dry storage (~42 weeks), and storage in nuclease-free water (~6 weeks).

3. Resuspending and storing your oligos in TE buffer is the best way to ensure oligo stability.

Since we began synthesizing them in 1987, IDT has continually studied the stability of our oligos under various storage temperatures and mediums. In all our studies, we have seen that oligos stored in a buffered solution are the most stable for the longest periods of time.

That is why we recommend you resuspend and store your DNA oligos in our TE buffer, IDTE (1X TE Solution; 10 mM Tris, 0.1 mM EDTA) rather than nuclease-free water. Tris [tris(hydroxymethyl)aminomethane] helps to maintain a constant pH, while EDTA (ethylenediaminetetraacetic acid) acts as chelating agent, preventing nuclease digestion of DNA. You can obtain IDTE in a variety of volumes, and you can select solutions at pH 7.5 or 8.0.

4. “Dry” oligos may not be so dry.

There is always some moisture present in a dry oligo, albeit a very small amount. And this moisture can cause DNA damage over time. That is why freezing your oligos is the best option for long-term storage. If you do choose to store your oligos at room temperature, resuspending them in TE buffer will provide a more stable environment than storing them dry.

Note: We ship most of our oligos dry, but the short shipping time will not jeopardize oligo stability. As mentioned earlier, dry oligos remain stable for over 40 weeks when stored at 37°C (98°F), which represents a “worst case scenario” shipping condition.

5. Freezing and thawing your oligos will have minimal impact on oligo stability.

It is not uncommon for some researchers to repeatedly freeze, then thaw their oligos while working in the lab. To address this, we functionally tested oligo stability after multiple freeze-­thaw cycles, using either nuclease-free water or IDTE Buffer as resuspension liquids (Figure 2). In our experiments, we found that 30 such cycles had no significant impact on oligo stability or function. This was true for both resuspension liquids tested (nuclease-free water and IDTE).

Figure 2. Oligo stability is maintained after 30 freeze-thaw cycles. A standard scale IDT PrimeTime® qPCR Assay containing oligonucleotide primers and probes was hydrated in IDTE Buffer to 40X. The tube was frozen (–20°C) and thawed 30 times. At (A) 0 and (B) 30 freeze-thaw cycles, an aliquot of the assay was run against a validated universal human reference cDNA standard curve (0.005–50 ng) using TaqMan® Gene Expression Master Mix (Thermo Fisher). The oligo components of the assay were stable and functional for up to 30 freeze-thaw cycles, showing no impact on Cq value. (C) The PrimeTime qPCR Assay at 0.5 ng cDNA concentration is shown in a reference dye-normalized view (Rn).

For modified DNA oligos and for RNA oligos:

6. Oligos with modifications have similar stability characteristics as unmodified oligos, and therefore should be stored using the same guidelines.

While we have not extensively tested long term stability of our full catalog of modifications using different storage conditions, we do expect most modified oligos to possess similar storage characteristics as unmodified oligos. Freezing is optimal when storing modified oligos for long durations, and IDTE Buffer will help preserve stability at temperatures above freezing.

Short term exposure to UV light, ambient lab light, or ozone should not affect function of fluorophore-modified oligos. However, when storing these oligos long term, we do recommend keeping them in the dark, regardless of storage medium, temperature, and duration; to prevent any negative impact on the fluorophore’s stability.

For additional information on the stability of specific modifications, please contact our applications support staff at applicationsupport@idtdna.com.

7. Preserving oligo stability is more difficult for RNA.

The chemical structure of RNA makes it inherently less stable than DNA. Further complicating RNA storage is the fact that RNases, which are present in human saliva, mucus, perspiration, and dead skin/hair cells, are often much more prevalent in standard laboratory conditions than DNases. Avoiding RNase contamination is crucial to maintaining RNA oligo stability, as even the slightest exposure to RNase can cause substantial degradation.

For short-term RNA storage, we suggest using our IDTE Buffer. If storing RNA oligos for long periods of time (months or years), RNA should be stored as an ethanol precipitate at –80°C [1].

The preceding facts and data were generated during an ongoing, multi-year longitudinal study of oligo stability. Our scientific application specialists are readily available to answer any further questions or to provide guidance on oligo handling and experimental setup. Contact them at applicationsupport@idtdna.com.